Human regulatory CD8+CD28- T cells (Ts) generated in vitro were demonstrated to suppress the activation and proliferation of T helper cells (Th) induced by allogeneic cells. This effect requires cell-to-cell contact, is antigen-specific, and results in Th anergy. To study the population of CD8+CD28- T cells present in vivo, flow cytometry was performed on whole blood specimens obtained from 25 heart transplant recipients and 12 normal controls. A significant expansion of CD8+CD28- T cells was found in transplant recipients as compared with normal individuals (p = 0.005). Expression of CD38, human leukocyte antigen-DR, and perforin positive cells within the CD8+CD28- subset was significantly higher in transplant patients than in normal controls, yet there was no correlation between the expression of these markers and acute rejection. Expression of the CD27 marker, however, was significantly higher within CD8+CD28- T cells from patients without rejection as compared with patients in rejection (p = 0.005), indicating that the memory-like CD8+CD28-CD27+ T-cell subset comprises regulatory cells, which play a protective role for the graft. CD8+CD28- T cells isolated from transplant patients did not display cytotoxic activity against donor cells and showed high expression of the killing inhibitory receptor CD94. This study identifies the phenotypic changes that occur in patients with heart transplants and opens new avenues for the induction of specific immunosuppression in transplantation.